The present invention concerns a device for and process of preparing volume flows of liquids in at least one passage of a chromatographic column, for analytical liquid measuring techniques.
Devices for preparing volume flows of liquids in capillary tubes are used in liquid chromatography, in particular high pressure liquid chromatography (HPLC). Depending on the internal diameters of the separation columns used, HPLC technology is divided into xe2x80x9cnormal bore technologyxe2x80x9d for separation columns with internal diameters in the range from approximately 3 to 5 mm, xe2x80x9cmicro bore technologyxe2x80x9d for separation columns with internal diameters in the range from approximately 1 to 2 mm, xe2x80x9ccapillary tube LC technologyxe2x80x9d for separation columns with internal diameters in the range from approximately 180 to 320 xcexcm, and xe2x80x9cnano LC technologyxe2x80x9d for separation columns with internal diameters of less than or equal to 100 xcexcm.
For these applications, pump systems are required to generate or transport liquids at extremely small flow rates or volume flows. These liquids must be transported with high reliability and precision under the effective high pressures in the range of approximately 400 bar.
Two different methods are known at present for transporting and preparing liquids having such small flow rates in capillary tube separation columns for liquid chromatography.
A first method is based on the use of injection pumps. Injection pumps are special single-piston pumps. In contrast to conventional piston pumps, in injection pumps the pistons do not move to and fro during analysis but a single piston stroke takes place. Thus the liquid in an injection pump is always in transport mode. The pump chamber must therefore be dimensioned sufficiently large that a single transport stroke is sufficient for complete separation analysis. The pump chamber is pressurized before analysis as the piston in the pump chamber is pressed forward. During separation analysis no more suction is performed. With this process, a volume flow is possible which is independent of the elasticities within the pump chamber. The elasticities in particular of the seals, drive mechanics and elasticity due to the compressibility of the solvent can be compensated accordingly. A further advantage of injection pump technology is the high precision and reproducibility of the achievable volume flows. As the pump chamber is constantly under pressure during the transport phase, the volume flow essentially depends only on the resolution of the drive and the seal of the total system.
Injection pump technology however only has a low flexibility with regard to the achievement of different analysis times and the use of different column diameters. Both the possible analysis time and the selection of separation column diameter are dependent and limited by the maximum stroke volume of the injection pump available at the time the analysis is to be performed. Furthermore with an injection pump only one high pressure gradient at a time can be achieved. This means that each solvent involved in the analysis requires its own high pressure injection pump.
A further possibility for generating and preparing liquid volume flows in capillary tubes, in particular in chromatographic separation columns, for analytical liquid separating technology is the use of conventional piston pumps suitable for xe2x80x9cnormal bore technologyxe2x80x9d in connection with the so-called splitter technology. Here suitable flow splitters are used in order to divide the total flow generated and transported by the pumps into at least two part flows, an excess flow in an excess path and a working flow in a working path. The working flow required in the separation column is adjusted and provided by means of restrictors, i.e., by hydraulic resistances arranged in the excess path. The flow splitters and in particular the hydraulic resistances are usually constructed from xe2x80x9cfused silica capillary tubesxe2x80x9d with small internal diameters. The length and internal diameter of these elements determine the flow resistance. The total flow rate is divided according to the resistance ratios, where normally the smaller part flows through the separation column.
One advantage of this technology is the low production cost because the splitters and the hydraulic resistances can be produced by the users. The extremely small volumes inside the flow splitters or hydraulic resistances are advantageous here.
One particular disadvantage of the conventional splitter technology however is that the user receives no information on the amount of volume flow passing through the separation column during separation analysis. Therefore the volume flow must be measured in a complex manner with mini-injections using stop watches in order to be able to operate the separation columns efficiently. Furthermore even the smallest changes in flow resistance caused, for example, by a contaminated separation column frit, lead to a considerable change in the column flow, with the result of a correspondingly large retention time shift. In order to alleviate this effect, a hydraulic pre-resistance is sometimes inserted in the working path before the separation column. Thus with approximately the same pressure reductions in the separation column and the pre-resistance, the influence of a blocked separation column frit on the column flow is approximately halved. The use of such pre-resistances however means that only half the pump pressure is available for separation analysis in the separation column.
An object of the invention is to provide a new and improved device for and a process of providing volume flows of liquids in capillary tubes for analytical liquid metrology to achieve an essentially constant working flow volume of the liquid being analyzed independent of back-pressure conditions.
This task is solved by the features of the claims, in particular in that the device has at least one working sensor and one control device to regulate the working flow rate and/or pressure in the liquid working path, where the control device is coupled to the working sensor and a means for changing the working flow rate. As a result, the working flow, i.e., the volume of liquid flowing through the capillary tube, can be kept essentially constant as a function of the pressure and/or volume conditions in the working path which change for example as a result of disturbance variables.
Suitably the means for changing the working flow volume is formed with a hydraulic resistance, in particular a nozzle. This allows smooth reproducible volume flows.
It is of particular advantage for the hydraulic resistance to have a flow resistance, in particular, to be continuously variable. Thus advantageously, variable hydraulic resistance values or xe2x80x9crestrictionsxe2x80x9d can be set with a single hydraulic resistance. Depending on the pressure and/or volume flow conditions changing in the working path, by corresponding change of the flow resistance, the working flow volume and/or working pressure can be held constant. One particular advantage in using variable hydraulic resistances is that both a volume flow and a pressure control are possible in the working path. With pressure control, the pressure in the working path can be held constant irrespective of the solvent used. Constant pressure in the working path is always advantageous if the solvent in the working path must be changed quickly without allowing the working pressure to become too high. With maximum flow rates, constant pressure in the working path allows a considerable extension of the life of the delicate parts contained in the working path, for example the separation column.
A further advantage in using variable hydraulic resistances is that when correspondingly dimensioned, a very great range of achievable flow rates is possible. If an extremely small flow resistance is used, i.e., with high flow rates through the hydraulic resistance, it is possible to flush the entire device including a degasification unit with high flow rates normally used in xe2x80x9cnormal bore technologyxe2x80x9d. This also allows correspondingly shorter flushing times. If a very high to infinite flow resistance is set, i.e., the volume flow through the hydraulic resistance tends towards zero, it is possible to use the device or separating system in a non-split mode. In the non-split mode, operation is possible which corresponds to that with a conventional piston pump.
A further advantage is that the total flow generated and transported by the transport device can be selected almost at will. A high flow rate allows a fast exchange of a dead volume between (1) a mixing point in the transport device formed for example as a pump, and (2) the capillary tube, which can be formed as a separation column. A high flow rate, at low pressure gradient operation, allows fast solvent change, i.e., faster gradients or short analysis times. In contrast when small total flow rates are set, large solvent quantities can be saved. Saving large solvent quantities is advantageous with regard to both environmental and cost aspects.
A further advantage of variable hydraulic resistances is that they can be formed as damping elements. When conventional piston pumps are used, on reversal of the piston short pressure impulses or pressure interruptions can occur. These impulses or interruptions can be compensated by suitably changing the hydraulic resistance, for example by brief closure thereof, with a controller. The pressure and/or volume flow fluctuations in the working path can thus be avoided without additional capacitative constructional elements.
As an alternative or in addition to the variable hydraulic resistances, the hydraulic resistance can also be formed with one or preferably plural fixed resistances which can be connected individually or in groups, preferably by a switching device, e.g., an electrically controlled valve. This valve can allow automatic connection of one or plural fixed resistances within an analysis frequency. The automatic connection can, for example, include a further control device and a further sensor, in particular a pressure sensor, allocated for example to the transport device or the total flow, as a function of the changing pressure conditions.
It is of particular advantage if the hydraulic resistance is arranged in the overflow path. Thus the required extremely small flow rates in the working path can be achieved flexibly at low cost with the required precision even under high pressures.
Alternatively or in combination with the use of hydraulic resistances, the working flow can be modified by a structure for changing the total flow, preferably with a transport device for transporting the total flow. If for example the resistance in the working path rises due to a blocked column inlet frit, the working flow in the working path is reduced accordingly. The total flow transported through the transport device can now be increased accordingly using the control device so that the working flow essentially remains constant.
Advantageously in this design, hydraulic resistances with modifiable flow cross sections and/or several hydraulic resistances with fixed resistances can also be used. In this way the total flow transported through the transport device can be varied within a narrower band.
Advantageously the device has one hydraulic resistance in the working path and one hydraulic resistance in the excess path. The hydraulic resistances are formed such that the working flow and the excess flow have different volumes. The working sensor includes at least one pressure sensor and a means for forming a pressure difference between pressures in the working path and excess path. Thus alone or in combination with other advantageous designs of the device, an essentially constant working flow volume can be achieved.
The volumetric measurement of the flow rates or volume flows necessary for liquid chromatography using capillary tube separation columns is extremely complex and difficult. This results in particular from the different physical properties of the different devices used and the occasionally very high pressures which can influence the measurement result. Volumetric flow meters for precise measurement of extremely small flow rates of liquids with different physical properties, for example with different compositions and/or concentrations, in capillary tubes for liquid measurement technology are not yet known. For these reasons, the working sensor is advantageously formed as a volumetric flow meter. A particularly advantageous volumetric flow meter for detecting the volumetric flow through the capillary tube can be achieved by forming the working sensor so it includes at least two detectors for detecting at least one gas bubble contained in the column flow. A time interval detector measures the run time differences of the gas bubbles passing the detectors.
It is known that mass flow sensors can detect minimum volume flows of liquids in capillary tubes. Such mass flow sensors, however, are always calibrated for only a single liquid or a single solvent and inherently function on a mass-selective basis. This means that the measurement result depends on the solvent used in each case. Suitable calibration of such mass flow meters for the different solvents used in liquid chromatography was previously either not possible or possible only with considerable expense.
According to a further particularly advantageous structure of the device, a hydraulic working resistance is arranged in the working path. Upstream of the hydraulic working resistance, a buffer device receives the liquids required for at least one calibration cycle. At least one pressure sensor measures the pressure or pressure drop over the hydraulic working resistance so that calibration of the working sensor is possible. The measurement value of the working sensor can be influenced by changing the physical properties of the liquids. The working sensor is preferably formed as a mass flow meter.
The device according to the invention enables simple and precise calibration of such mass flow meters, with regard to various solvents used during separation analysis. This results from the combination of a volume flow control and a pressure control. The volume flow and pressure controllers control a suitable means for modifying the working flow, a variable hydraulic resistance that is preferably continuously modifiable.
If the device contains a working sensor calibrated in this manner, essentially constant volume flows can be achieved in the working path even for liquids with physical properties which change, for example in gradient operation. In addition the user receives precise information on what volume flow is passing through the capillary tubes of the separation column which form a hydraulic resistance during separation analysis in the working path.
The invention also concerns a process of preparing volume flows of liquids in capillary tubes, in particular in chromatographic separation columns for analytical liquid metrology. A transport device transports a total flow which is divided by a flow splitter into an excess flow in an excess path and a working flow in a working path. A control device coupled with a working sensor and a means for changing the working flow volume govern the working flow volume and/or the pressure in the working path. Thus, independently of the back-pressure conditions, an essentially constant working flow volume can be achieved.
This process can be used particularly advantageously to calibrate working sensors in which a measurement value can be influenced by changing physical properties of the liquids. Such working sensors include a mass flow sensor in the working path. The sensor includes a hydraulic working resistance responsive to liquid flowing from a buffer device that receives the liquids required for at least one calibration cycle. At least one pressure sensor measures the pressure or pressure drop over the hydraulic working resistance. In a first step, a first liquid is transported in the working path and the volume flow of this first liquid is held essentially constant. The pressure sensor detects the pressure or pressure drop over the hydraulic resistance. In a second stage, the pressure or pressure drop over the hydraulic working resistance that was determined in the first stage is held essentially constant and a second liquid with physical properties different from those of the first liquid is transported in and through the working path at least until a working sensor can measure a corresponding measurement value. This measurement value allows calibration of the working sensor with regard to the second liquid.
Suitably the second step is performed several times in succession. In each case, the second step is performed with a third or last liquid which has physical properties different from the properties of the liquid previously transported through the working paths. Thus the working sensor can be calibrated simply and precisely for the liquid gradients required in liquid chromatography, for example HPLC technology.
Advantageously, the working sensor is calibrated by performing the first and second stages several times in succession, in each case with changed total pressures. Thus the working sensor can simply be calibrated precisely for different total pressures.
Suitably before the first stage and/or after the second or last stage of a calibration cycle, the working path is flushed with a first liquid. During flushing with the first liquid, the pressure or pressure drop over the hydraulic resistance is held essentially constant. Preferably during calibration cycle flushing, a greater total flow is transported or a higher total pressure is set. These measures allow fast and thorough flushing of the working path without the working pressure becoming excessively high. Consequently at maximum flow rates a considerable extension of the life of sensitive parts contained in the working path, for example, the separation column, is achieved.
The invention also concerns the use of a device with the features described above in capillary tube liquid chromatography.
The above measures contribute both individually and in combination to a particularly precise and reproducible separation result.